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(a) Schematic representation of fDET. A single-tube multiplex <t>PCR</t> combined with capillary electrophoresis–based fragment analysis enables simultaneous detection of up to 18 target genes. During early PCR cycles, gene-specific forward primers with a poly-dC extension add a poly-dC sequence to the 5′ end of target amplicons. After depletion of the low-concentration forward primers, a FAM-labeled poly-dC primer and gene-specific reverse primers amplify the tailed fragments, resulting in fluorescently labeled amplicons. (b) Optimized fDET result using a positive control consisting of pooled <t>genomic</t> <t>DNA</t> extracted from plants harboring various transgenes. (c)-(e) Examples of identification of transgenes by fDET. (c) GFP reporter line, (d) dSpm transposon insertion line from the SLAT collection, and (e) Kazusa T-DNA insertion line. (f)-(h) Sensitivity of fDET. fDET detect contamination as low as 1%. (f) Col (non-transgenic), (g) Col spiked with 3% T-DNA insertion line, (h) Col spiked with 1% T-DNA insertion line. The spiked line is identical to that shown in (e). (i) Schematic representation of DET. Four multiplex PCR reactions followed by agarose gel electrophoresis–based fragment analysis detect up to 11 target genes. (j)-(M) Examples of transgene identification by DET. (J) Det-1 primer mix ( NPTII, HPT and BAR ), (K) Det-2 primer mix ( GFP, GUS and LUC ), (L) Det-3 primer mix (35S promoter and NOS promoter), and (m) Det-4 primer mix ( NOS terminator and Cas9 ). M: marker, PC: positive control. Analyzed samples are listed in Supplemental Table S3.
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(a) Schematic representation of fDET. A single-tube multiplex <t>PCR</t> combined with capillary electrophoresis–based fragment analysis enables simultaneous detection of up to 18 target genes. During early PCR cycles, gene-specific forward primers with a poly-dC extension add a poly-dC sequence to the 5′ end of target amplicons. After depletion of the low-concentration forward primers, a FAM-labeled poly-dC primer and gene-specific reverse primers amplify the tailed fragments, resulting in fluorescently labeled amplicons. (b) Optimized fDET result using a positive control consisting of pooled <t>genomic</t> <t>DNA</t> extracted from plants harboring various transgenes. (c)-(e) Examples of identification of transgenes by fDET. (c) GFP reporter line, (d) dSpm transposon insertion line from the SLAT collection, and (e) Kazusa T-DNA insertion line. (f)-(h) Sensitivity of fDET. fDET detect contamination as low as 1%. (f) Col (non-transgenic), (g) Col spiked with 3% T-DNA insertion line, (h) Col spiked with 1% T-DNA insertion line. The spiked line is identical to that shown in (e). (i) Schematic representation of DET. Four multiplex PCR reactions followed by agarose gel electrophoresis–based fragment analysis detect up to 11 target genes. (j)-(M) Examples of transgene identification by DET. (J) Det-1 primer mix ( NPTII, HPT and BAR ), (K) Det-2 primer mix ( GFP, GUS and LUC ), (L) Det-3 primer mix (35S promoter and NOS promoter), and (m) Det-4 primer mix ( NOS terminator and Cas9 ). M: marker, PC: positive control. Analyzed samples are listed in Supplemental Table S3.
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(a) Schematic representation of fDET. A single-tube multiplex <t>PCR</t> combined with capillary electrophoresis–based fragment analysis enables simultaneous detection of up to 18 target genes. During early PCR cycles, gene-specific forward primers with a poly-dC extension add a poly-dC sequence to the 5′ end of target amplicons. After depletion of the low-concentration forward primers, a FAM-labeled poly-dC primer and gene-specific reverse primers amplify the tailed fragments, resulting in fluorescently labeled amplicons. (b) Optimized fDET result using a positive control consisting of pooled <t>genomic</t> <t>DNA</t> extracted from plants harboring various transgenes. (c)-(e) Examples of identification of transgenes by fDET. (c) GFP reporter line, (d) dSpm transposon insertion line from the SLAT collection, and (e) Kazusa T-DNA insertion line. (f)-(h) Sensitivity of fDET. fDET detect contamination as low as 1%. (f) Col (non-transgenic), (g) Col spiked with 3% T-DNA insertion line, (h) Col spiked with 1% T-DNA insertion line. The spiked line is identical to that shown in (e). (i) Schematic representation of DET. Four multiplex PCR reactions followed by agarose gel electrophoresis–based fragment analysis detect up to 11 target genes. (j)-(M) Examples of transgene identification by DET. (J) Det-1 primer mix ( NPTII, HPT and BAR ), (K) Det-2 primer mix ( GFP, GUS and LUC ), (L) Det-3 primer mix (35S promoter and NOS promoter), and (m) Det-4 primer mix ( NOS terminator and Cas9 ). M: marker, PC: positive control. Analyzed samples are listed in Supplemental Table S3.
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(a) Schematic representation of fDET. A single-tube multiplex PCR combined with capillary electrophoresis–based fragment analysis enables simultaneous detection of up to 18 target genes. During early PCR cycles, gene-specific forward primers with a poly-dC extension add a poly-dC sequence to the 5′ end of target amplicons. After depletion of the low-concentration forward primers, a FAM-labeled poly-dC primer and gene-specific reverse primers amplify the tailed fragments, resulting in fluorescently labeled amplicons. (b) Optimized fDET result using a positive control consisting of pooled genomic DNA extracted from plants harboring various transgenes. (c)-(e) Examples of identification of transgenes by fDET. (c) GFP reporter line, (d) dSpm transposon insertion line from the SLAT collection, and (e) Kazusa T-DNA insertion line. (f)-(h) Sensitivity of fDET. fDET detect contamination as low as 1%. (f) Col (non-transgenic), (g) Col spiked with 3% T-DNA insertion line, (h) Col spiked with 1% T-DNA insertion line. The spiked line is identical to that shown in (e). (i) Schematic representation of DET. Four multiplex PCR reactions followed by agarose gel electrophoresis–based fragment analysis detect up to 11 target genes. (j)-(M) Examples of transgene identification by DET. (J) Det-1 primer mix ( NPTII, HPT and BAR ), (K) Det-2 primer mix ( GFP, GUS and LUC ), (L) Det-3 primer mix (35S promoter and NOS promoter), and (m) Det-4 primer mix ( NOS terminator and Cas9 ). M: marker, PC: positive control. Analyzed samples are listed in Supplemental Table S3.

Journal: bioRxiv

Article Title: Multiplex PCR based Detection Methods of Common Plant Transgenes

doi: 10.64898/2026.04.23.720246

Figure Lengend Snippet: (a) Schematic representation of fDET. A single-tube multiplex PCR combined with capillary electrophoresis–based fragment analysis enables simultaneous detection of up to 18 target genes. During early PCR cycles, gene-specific forward primers with a poly-dC extension add a poly-dC sequence to the 5′ end of target amplicons. After depletion of the low-concentration forward primers, a FAM-labeled poly-dC primer and gene-specific reverse primers amplify the tailed fragments, resulting in fluorescently labeled amplicons. (b) Optimized fDET result using a positive control consisting of pooled genomic DNA extracted from plants harboring various transgenes. (c)-(e) Examples of identification of transgenes by fDET. (c) GFP reporter line, (d) dSpm transposon insertion line from the SLAT collection, and (e) Kazusa T-DNA insertion line. (f)-(h) Sensitivity of fDET. fDET detect contamination as low as 1%. (f) Col (non-transgenic), (g) Col spiked with 3% T-DNA insertion line, (h) Col spiked with 1% T-DNA insertion line. The spiked line is identical to that shown in (e). (i) Schematic representation of DET. Four multiplex PCR reactions followed by agarose gel electrophoresis–based fragment analysis detect up to 11 target genes. (j)-(M) Examples of transgene identification by DET. (J) Det-1 primer mix ( NPTII, HPT and BAR ), (K) Det-2 primer mix ( GFP, GUS and LUC ), (L) Det-3 primer mix (35S promoter and NOS promoter), and (m) Det-4 primer mix ( NOS terminator and Cas9 ). M: marker, PC: positive control. Analyzed samples are listed in Supplemental Table S3.

Article Snippet: Between 1 to 100 ng of genomic DNA was used for multiplex PCR reactions using the QIAGEN Multiplex PCR Kit (QIAGEN), 1 μL of the primer mix in a total reaction volume of 10 μL.

Techniques: Multiplex Assay, Electrophoresis, Sequencing, Concentration Assay, Labeling, Positive Control, Transgenic Assay, Agarose Gel Electrophoresis, Marker